DISCLAIMER: This document does not meet the current format guidelines of. the Graduate School at. The University of Texas at Austin.

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1 DISCLAIMER: This document does not meet the current format guidelines of the Graduate School at The University of Texas at Austin. It has been published for informational use only.

2 Copyright by Michael Anthony Sandoval 2012

3 The Thesis Committee for Michael Anthony Sandoval Certifies that this is the approved version of the following thesis: Targeted Nanoparticle Formulation for a Poorly Water Soluble Gemcitabine Derivative and its In Vivo and In Vitro Anti-tumor Activity APPROVED BY SUPERVISING COMMITTEE: Supervisor: Zhengong Cui Janet Walkow Robert O. Williams III

4 Targeted Nanoparticle Formulation for a Poorly Water Soluble Gemcitabine Derivative and its In Vivo and In Vitro Anti-tumor Activity by Michael Anthony Sandoval, B.S.; B.S. Thesis Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Master of Science in Pharmacy The University of Texas at Austin August 2012

5 Dedication To my loving parents, who have always supported my educational and personal goals. To my professional development mentors, whose vision and guidance provided personal growth and challenged me to be an innovator in teaching, research, and education. To the College of Pharmacy faculty for their efforts and support to provide an enriching and thorough pharmaceutical education. To all my Pharm.D students, who held me to the highest standards of teaching, knowledge, and compassion. And finally, to my closest friends and loved one for their support, patience, and time they have sacrificed to make this all possible.

6 Acknowledgements In joy, I would like to extend my deepest gratitude and sincerest appreciation to my closest friends and family, for their patience, support, and love, all of which made my experiences at The University of Texas at Austin productive and enjoyable. Most notably, I would like to thank present and former researchers in Dr. Cui s lab: Mr. Amit Kumar, Dr. Nijaporn Yanasarn, Dr. Dharmika Lansakara, Ms. Letty Rodriguez, Ms. Xinran Li, Dr. Melisande Holzer, Dr. Woongye Chung, and Dr. Rebecca De Angel. I would like to specifically highlight Dr. Brian R. Sloat s contribution towards my success in graduate school. He was kind, patient, and most of all willing to train/mentor me as well as everyone else in the lab. I would also like to acknowledge God, my lord and savior, who has always guided me through the walks of life, providing means for intellectual apprehension, courage to stand up for what is right, and the reason to love all whom I encounter. I am especially grateful for the other graduate students in the pharmacy program, for who they are and the enthusiasm they bring to the scientific community. I owe a special thanks to my supervisor, Dr. Zhengrong Cui, for his high expectations, and intellectual and personal guidance he provided throughout my graduate education. I would like to thank Dr. Janet Walkow, for her unprecedented business aptitude, as well as her admirable people skills and practical knowledge in translational research. She is really someone special and eager to make positive impacts on society. Moreover, her advice throughout my graduate education has always been kind and helpful. As a graduate student at UT, I had the privilege to sit, listen, and learn from the finest professors in the field I would like to give my upmost gratitude and respect to Dr. Robert Williams, Dr. James McGinity, Dr. Salomon Stavchansky, Dr. Hugh Smyth, Dr. v

7 Jason McConville, and Dr. Maria Croyle for all the hard work and passion they invest into teaching and mentoring. Their expertise in the pharmacy field made my learning experiences invaluable. I look up to them all. I would like to especially thank Dr. Salomon Stavchansky, as he was especially helpful in enhancing and fine-tuning my experiences as a teaching assistant for the professional pharmacy program. His ability to connect with students, honest opinion, and breathe of personal experiences made him quite inspirational and a delight to work with. Last but definitely not least, I would like to express thanks to the supporting staff in the College of Pharmacy: Ms. Yolanda Abasta, a sweet and lively women, always was tremendously helpful and a breath of fresh of air. Mr. Jay Hamman and Mr. Kamran Ziai, thank you so much for the genuine kindness and sincere support. vi

8 Abstract Targeted Nanoparticle Formulation for a Poorly Water Soluble Gemcitabine Derivative and its In Vivo and In Vitro Anti-tumor Activity Michael Anthony Sandoval, M.S.Phr The University of Texas at Austin, 2012 Supervisor: Zhengrong Cui Cancer is a collection of over one hundred different types of diseases and is responsible for the leading cause of death in the United States. More strikingly, cancer mortality rates have remained relatively unchanged for the past several decades, indicating significant clinical demand for improved cancer therapy. Gemcitabine, known clinically as Gemzar, is used to treat a variety of human cancers, however, clinical efficacy is modest due to it s brief blood circulation, rapid clearance, manifestation of tumor-drug resistance, and lack of drug specificity. This thesis sought to develop a solid lipid nanoparticle-based platform to passively and actively target a gemcitabine lipophilic derivative, 4-(N)-stearoyl gemcitabine, into tumor cells over-expressing epidermal growth factor receptor (EGFR) after intravenous injection. Considering gemcitabine is hydrophilic and the core of the nanoparticle is solid (hydrophobic), we lipophilized gemcitabine by conjugating a stearoyl group to its N-terminus to form 4-(N)-stearoyl gemcitabine. Second, we incorporated stearoyl gemcitabine into lecithin-based nanoparticles. The nanoparticle formulation was prepared from lecithin/glyceryl vii

9 monostearate-in-water emulsions. Third, we grafted the gemcitabine nanoparticles with polyethylene glycol chains with reactive end groups that are capable of conjugating with a targeting moiety on the surface to actively target tumors that over-express EGFR. Taken together, the overall objective of the research presented in this thesis is to develop, characterize, and evaluate the anti-tumor performance in vitro as well as in mice against both human and mouse tumor models.. viii

10 Table of Contents List of Tables...xii List of Figures...xiii Chapter 1: General Introduction Motivation: Statistics and Significance Cancer and Chemotherapy Gemcitabine as a Chemotherapeutic Drug Gemcitabine and its Clinical Limitations Cancer Nanotechnology Cancer Drug Targeting: The Epidermal Growth Factor Receptor References...11 Chapter 2: In Vivo and In Vitro Anti-tumor Activities of a Gemcitabine Derivative Carried by Nanoparticles Abstract Introduction Materials and Methods Chemicals and Cell-lines Synthesis of 4-(N)-stearoyl gemcitabine Incorporation of 4-(N)-stearoyl gemcitabine into nanoparticles Transmission Electron Microscopy (TEM) Gel Permeation Chromatography (GPC) In Vitro Release of GemC18 from GemC18-NPs and PEG- GemC18- NPs HPLC Uptake of Nanoparticles by Tumor Cells in Culture In Vitro Cytotoxicity Assay In Vivo Tumor Treatment Studies In Vivo and Ex Vivo Fluorescence Imaging Statistics...24 ix

11 2.4 Results and Discussion Preparation and Characterization of Stearoyl Gemcitabine Incorporated Solid Lipid Nanoparticles Uptake of Nanoparticles by Tumor Cells in Culture Evaluation of the Cytotoxicity of the GemC18-NPs and PEGylated GemC18-NPs in Tumor Cell in Culture Biodistribution of GemC18-NPs and PEG-GemC18-NPs in Tumorbearing Mice Evaluation of the Anti-tumor Activity of the GemC18-NPs in Mice with Pre-grafted Tumors Comparison of the In Vivo Anti-tumor Activities of the PEGylated and un-pegylated Gemcitabine Nanoparticles The PEGylated GemC18-NPs Were More Effective than the GemC18-in-Tween 20 Micelles in Controlled Tumor Growth Conclusions References...36 Chapter 3: EGFR-targeted Stearoyl Gemcitabine Nanoparticles Show Enhanced Anti-tumor Activity Abstract Introduction Materials and Methods Materials and Cell-lines Preparation and Characterization of EGFR-targeted Stearoyl Gemcitabine Nanoparticles In Vitro Cellular Uptake Assay Fluoroscence Microscopy for the Detection of the Uptake of Nanoparticles Flow Cytometry In Vitro Cytotoxicity Evaluation of the In Vivo Anto-tumor Activity of the EGF- GemC18-NPs Ex Vivo Imaaging Using IVIS Imaging Histology...47 x

12 3.3.10Statistical Analysis Results and Discussion Preparation and Characterization of EGF-GemC18-NPs and OVA- GemC18-NPs In Vitro Uptake of the EGF-GemC18-NPs by Tumor Cells Expressing Different Levels of EGFR Correlation of the In Vitro Cytotoxicity of the EGF-GemC18-NPs with the Density of EGFR on Tumor Cells The EGFR-targeting GemC18-NPs More Effectively Controlled the Growth of Pre-established EGFR-over-expressing Tumors in Mice The EGF on the EGF-GemC18-NPs Increased the Accumulation of the Nanoparticles in Tumors in Mice Conclusions References...57 Chapter 4: Anti-tumor Efficacy of Oral 4-(N)-Stearoyl Gemcitabine Nanoparticles Abstract Introduction Materials and Methods Chemicals and Cell-lines Preparation of 4-(N)-Stearoyl Gemcitabine Nanoparticles Stability of 4-(N)-Stearoyl Gemcitabine Nanoparticles In Vitro Release in Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) In vivo Anti-tumor Efficacy Study Plasma Pharmacokinetics Results and Discussion Conclusions References...71 Tables and Figures...73 Bibliography...95 xi

13 List of Tables Table 1: Characterization of nanoparticles. Data shown are mean ± SD (n 3):.73 Table 2: Gemcitabine pharmacokinetic parameters in plasma after intravenous (i.v) or oral (p.o) administration of GemC18-NPs in healthy BALB/c mice: xii

14 List of Figures Figure 2.1: Preparation and characterization of GemC18-NPs. (A) In GPC, GemC18- free-nps and GemC18-NPs eluted about two fractions earlier than GemC18 in Tween 20 micelles ( )..The concentration of the GemC18 in the micelles and GemC18-NPs was 100 μg/ml. (B) Gel permeation chromatographs of GemC18-NPs prepared with 0, 0.1, 0.5, 1, 2.5, and 5 mg/ml of GemC18. In A and B, gemcitabine was measured at 248 nm. (C) TEM micrograph of the GemC18-NPs (with 5 mg/ml of GemC18). (D) Chromatographs of GemC18-NPs ( ) and PEGylated GemC18-NPs (Δ) prepared with 5 mg/ml of GemC18. (E) The size and zeta potential of the GemC18-NPs and the PEG-GemC18-NPs. (F) The dynamic light scattering spectra of the GemC18-in-Tween 20 micelles (left), GemC18-NPs, and PEG-GemC18- NPs (far right) overlaid. (G) The release of the GemC18 from the GemC18- NPs ( ) or PEG-GemC18-NPs (Δ). (I) The size of the GemC18-NPs and PEG-GemC18-NPs after 30 min of incubation at 37 C in FBS in normal saline. Except in C and F, all data presented were the mean from at least 3 independent determinations. Standard deviations were not included in some figures for clarity xiii

15 Figure 2.2: The uptake of GemC18-NPs by TC-1 tumor cells in culture. (A) Fluorescence micrographs. Cells were incubated with fluorescein-labeled GemC18-NPs for 6 h at 37 C or 4 C and observed under a bright-field microscope (left panel) or a fluorescence microscope (right panel). Photos were taken at 20 magnification. (B) Comparison of the uptakes of PEGylated and un-pegylated GemC18-NPs. *p < 0.001, PEG-GemC18- NPs vs. GemC18-NPs at 37 C Figure 2.3: GemC18-NPs were cytotoxic to tumor cells in culture. (A) The IC 50 values of gemcitabine, GemC18-NPs, and PEG-GemC18-NPs in TC-1 and BxPC-3 cells. Cells were incubated with gemcitabine HCl or nanoparticles for 48 h. *For both cell lines, p < 0.05, Gemcitabine vs. GemC18-NPs. (B) It took the GemC18-NPs a longer time than thegemcitabine HCl to kill tumor cells. TC-1 cells were incubated with gemcitabine HCl or GemC18-NPs at 28.7 nm for 24 or 48 h, and the % of surviving cells was determined. Data are mean ± S.D. (n = 3 4)...78 xiv

16 Figure 2.4: In vivo and ex vivo imaging of GemC18-NPs and PEG-GemC18-NPs. (A) IVIS images of athymic mice 24 h after injection of fluorescein-labeled GemC18-NPs or PEG-GemC18-NPs. (B) Relative fluorescence intensity values in BxPC-3 tumors (circular ROI in A). a p = , GemC18-NPs vs. PEG-GemC18-NPs. (C) Tissue distribution of fluorescein-labeled GemC18- NPs and PEG-GemC18-NPs 24 h after injection. b GemC18-NPs vs. PEG- GemC18-NPs, p = 0.003, 0.021, and for blood, liver, and spleen, respectively...79 Figure 2.5: In vivo anti-tumor activity of GemC18-NPs against BxPC-3 tumors in athymic mice. (A) BxPC-3 tumor growth curves. Tumor cells were seeded on day 0, and mice were i.v. injected on days 6 and 19. (B) Average weight of BxPC-3 tumor-bearing mice after different treatments. *p = (ANOVA on week 3)...80 xv

17 Figure 2.6: Comparison of the in vivo anti-tumor activities of GemC18-NPs and PEGylated GemC18-NPs. (A) TC-1 tumors in C57BL/6 mice. Mice (n = 5 7) were injected (i.v.) with GemC18-NPs or PEG-GemC18-NPs once (1 mg GemC18 per mouse). (B) BxPC-3 tumors in athymic mice. Mice (n = 5) were injected (i.v.) with GemC18-NPs or PEG-GemC18-NPs 3 times (days 0, 12, and 21). In A and B, tumor sizes were reported starting from the day of the injection of the nanoparticles. Data shown are mean ± S.E.M. Statistical analysis did not reveal any differences between the GemC18-NPs and PEG-GemC18-NPs in A B Figure 2.7: Healthy C57BL/6 mice (n = 3) were injected via the tail vein with GemC18- NPs (1 mg of GemC18/mouse) or mg of gemcitabine HCl in sterile mannitol (5%). As controls, mice were either injected (i.v.) with polyriboinosinic-polyribocytidylic acid (poly(i:c) or pi:c, 50 μg/mouse (Sigma) as a positive control or sterile mannitol solution as a negative control. Twenty-four h (A) or 7 days (B) later, mice were euthanized to collect blood samples. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in the serum samples were determined using AST and ALT kits from Teco Diagnostics (Anaheim, CA). Data reported are mean ± S.D. in A, * p < , ** p = 0.008, Control vs. Gemcitabine..82 xvi

18 Figure 3.1: In vitro uptake of EGF-GemC18-NPs by tumor cells expressing different levels of EGFR. (A). MCF-7, MDA-MB-231, or MDA-MB-468 cells were incubated with fluorescein-labeled EGF-GemC18-NPs (EGF-NPs) or fluorescein-labeled OVA-GemC18-NPs (OVA-NPs) for 6 h, and the extent of nanoparticle uptake was determined by measuring the fluorescence intensity (*p = ; **p = 0.03). (B). The uptake of the EGF-GemC18- NPs or OVA-GemC18-NPs by MDA-MB-468 cells with (EGF+) or without pre-incubation of the cells with free EGF (***p = 0.009, EGF-NPs vs. EGF + EGF-NPs). The initial fluorescence intensity values of the EGF-NPs and the OVA-NPs were not different. Data shown are mean ± S.D. (n = 4 in A, and 3 in B) Figure 3.2: Flow cytometric and fluorescent microscopic analyses of the uptake of EGF-GemC18-NPs by tumor cells expressing different levels of EGFR. (A). Typical flow cytometric graphs of cells after 6 h of incubation with fluorescein-labeled EGF-GemC18-NPs (green, far right), fluoresceinlabeled OVA-GemC18-NPs (gray, middle), or sterile PBS (solid gray area). Experiment was repeated three times with similar results. (B). Fluorescent microscopic images of MDA-MB-468 and MCF-7 cells after 6 h of incubation with EGF-GemC18-NPs, OVA-GemC18-NPs, or sterile PBS (control). Cell nucleus was stained with DAPI (blue). Nanoparticles were labeled with fluorescein and shown in green 84 xvii

19 Figure 3.3: In vitro cytotoxicity of EGF-GemC18-NPs. (A). Percent of cells alive after 48 h of incubation with different concentration of GemC18 in EGF- GemC18-NPs or OVA-GemC18-NPs (n = 4). (B). Flow cytometric graphs of MDA-MB-468 cells after 24 h of incubation with EGF-GemC18-NPs or OVA-GemC18-NPs and stained with Annexin V and 7-AAD. Numbers in the quadrants are % of cells in early apoptosis (LR), late apoptosis (UR), and cell debris (UL). Experiment was repeated 3 times with similar results...86 Figure 3.4: Anti-tumor activity of EGF-GemC18-NPs in nude mice with pre-established MDA-MB-468 tumors. (A). Tumor growth curves. (B). Mouse survival curves (p = 0.053, EGF-GemC18-NPs vs. OVA-GemC18-NPs). In A and B, mice were dosed 11, 17, 28, and 37 days after tumor cell injection. (C). Tumor growth curves in mice used for immunohistology analysis. In A and C, *p < 0.05, EGF-GemC18-NPs vs. OVA-GemC18-NPs..87 Figure 3.5: Immunohistographs of MDA-MB-468 tumors after treatment with EGF- GemC18-NPs or OVA-GemC18-NPs. (A). Tumor tissues were staining with antibodies against BrdU, CD31, or caspase 3 (Cas 3). Scale bars = 100 μm. (B). The % of BrdU positive cells. (C). The % of caspase 3 positive cells xviii

20 Figure 3.6: Biodistribution of EGF-GemC18-NPs. (A). Ex vivo fluorescence IVIS images of MDA-MB-468 tumors and organs 24 h after injection (T = tumor, K = kidney, H = heart, and S = spleen). (B). A comparison of the normalized fluorescence intensity of EGF-GemC18-NPs or OVA-GemC18- NPs in tumors and different organs 24 h after injection (*, p = in tumors). (C). Fluorescence intensity in the blood of healthy C57BL/6 mice at different time points after i.v. injection of fluorescein-labeled nanoparticles. Data shown in B and C are mean ± S.D. from three replicates...89 Figure 3.7: At 28.3 mg/kg, gemcitabine hydrochloride was toxic to nude mice with MDA-MB-468 tumors. MDA-MB-468 tumor cells (1 107 cells/mouse) were mixed with BD Matrigel TM (50%:50%) and subcutaneously injected in the right flank of the mice on day 0. On day 11, mice were randomized and injected intravenously (i.v.) via the tail vein with 200 μl of gemcitabine hydrochloride (n = 5), OVA-GemC18-NPs in sterile mannitol (5%, w/v) (n = 7) or sterile mannitol alone as a negative control (n = 5). Injection was repeated on days 17, 28, and 37. The dose of the GemC18 was about mg per mouse per injection; the dose of the gemcitabine hydrochloride was mg per mouse per injection.. 90 Figure 4.1: Stability of GemC18-NPs after incubation in Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) xix

21 Figure 4.2: In vitro release of GemC18 from GemC18-NPs or GemC18-in Tween 20 micelles (GemC18-Micelles) in Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) Figure 4.3: In vivo anti-tumor activity of GemC18-NPs when given orally (p.o) and intravenously (i.v) against TC-1 tumors in C57BL/6 mice. (A) TC-1 tumor growth curve. (B) Mouse survival curve Figure 4.4: Plasma gemcitabine concentration (µg/ml) versus time (h) curves after GemC18-NPs were (A) intravenously (i.v) injected or (B) orally (p.o) gavaged into mice xx

22 Chapter 1: General Introduction 1.1 Motivation: Statistics and Significance Nearly 1.6 million cases of cancer will be diagnosed in year 2012, and will claim an estimated 577,190 lives in the United States [1]. As a result, cancer ranks as the second leading cause of death after heart disease, accounting for nearly one of every four deaths. Globally, the World Health Organization estimates an astonishing 7.6 million deaths from cancer to lead as the number one cause of death in Economically, the National Institute of Health projected the overall cost for cancer in the United States was $226.8 billion: $103.8 billion for direct medical costs and $123 billion for indirect mortality costs [1]. Interestingly, for the past several decades the scientific community has made exceptional progress towards the deeper understanding in cancer biology and advancing therapeutic options, however this has not translated into even distantly comparable advances in the clinic. If mortality rates of other chronic diseases (i.e. heart, cerebrovascular disease, and influenza) are compared to that of cancer from 1991 to 2006, its shown that while heart disease and other major chronic diseases decreased substantially during this period, the mortality rates of cancer remained relatively constant (~16% decrease) [1]. Taken together, proportional and additional research is needed to gain a better understanding of cancer and identify new therapeutic options to fight this disease. 1.2 Cancer and Chemotherapy Cancer is a collection of over one hundred different types of diseases and is responsible to consume as the second leading cause of death in the United States. The mechanism by which cancer develops and progresses is not completely understood, as 1

23 there are several distinct pathways this can happen. However, its widely accepted that cancer is thought to develop from a cell in which the normal mechanism for control of growth and proliferation is abnormally altered [2]. A large body of research has accumulated demonstrating the concept of cancer development is a multi-stage process that is genetically regulated in three stages. The first stage initiation, requires cell exposure to carcinogenic substances that produce genetic damage, and if not mended, results in irreversible cellular mutations and the potential to develop into influential populations of cancer cells [2]. Substances that may act like carcinogens can be chemicals, physical, and/or biological agents. In the second stage, known as promotion, carcinogens stimulate the newly initiated cancerous environment to grow and flourish. Additionally, age, gender, diet, growth factors, and chronic inflammation may act together or in sequence to promote the development of cancer. Scientific literature states that several years (~10-20 years) can pass before any clinically relevant symptoms manifest [1-3]. Torring et al. conducted a population-based study that investigated the relationship between diagnostic interval and mortality in 268 colorectal cancer patients. They found that in patients that present clinical symptoms of cancer, the risk of dying within three years decreased with diagnostic intervals up to five weeks and then increased (p = 0.002). Moreover, the American Cancer Society believes there is evidence to suggest that one-third of the involved cancer deaths in 2012 is directly correlated to physical inactivity and obesity. Meaning, innovative chemoprevention strategies can potentially aim to improve lifestyle and diet, thereby increasing the time at which cancer is detected, however more research needs to be conducted for further conclusions and recommendations. The third stage of cancer development is known as progression, which involves further genetic mutations leading to increase cellular proliferation, invasion, and development of metastases [2]. 2

24 It has been reported that approximately 85% of cancers develop solid tumors [4], and depending on the tumor biology, more than half of deaths result. A solid tumor may arise from four tissue types: epithelial, connective, lymphoid, and nerve tissues; and are classified as either benign or malignant. By definition, benign tumors are non-cancerous, localized, and rarely metastasize [2]. In contrast, malignant tumors are genetically unstable and have the ability to invade contiguous tissues and metastasize to the surroundings. The primary modality in the approach to cancer treatment is surgery for most localized, solid tumors. However, because most cancer patients have metastatic disease at time of diagnosis, localized therapies are often obsolete, failing to produce a cure. Therefore, the goals of systemic therapy using highly cytotoxic drugs, referred to as chemotherapy, are intended to treat primary solid tumors and any metastatic disease. The remnants of cancer therapy using chemotherapy can be traced back to the late 1940 s when Goodman and Gilman first administered nitrogen mustard to patients with lymphoma [3]. The fundamental medical objective of chemotherapy is to destroy and prevent cancer cells from multiplying and metastasizing. In general, chemotherapeutic drugs, with the exception of immunotherapeutics (not covered in this thesis) are efficacious by exerting their effects primarily to the mammalian cell cycle [5]. All tumor cells, as well as normal cells in the body, undergo the process of cell division and renewal, all of which involves the five-phase cell cycle: G0, G1, S, G2, and M. The G0 phase ( resting phase ) is the starting point of the cell cycle in which no division occurs. The actively reproducing phases (G1, S, G2, and M) are associated with ready division synthesis, pre-mitosis, and mitosis, respectively [5]. Depending on which phase(s) is altered or interfered, chemotherapeutic drugs are categorized into two groups: cell cycle specific and cell cycle non-specific [3,5]. Cell cycle specific drugs have the ability to 3

25 exert its affect on a particular phase of the cell cycle and not on the resting phase. Whereas, cell cycle non-specific drugs can attack cells at any phase of the cycle [5-6]. 1.3 Gemcitabine as a Chemotherapeutic Drug Gemcitabine is an anti-metabolite indicated to treat a variety of human cancers including breast, ovarian, and non small cell lung cancers, yet, gemcitabine has been clinically adopted as the first-line treatment for patients diagnosed with locally advanced or metastatic pancreatic cancer [7]. Gemcitabine is known commercially as Gemzar upon FDA approval in 2004, and originally marketed and distributed by Eli Lilly & Company. Due to Gemzar s recent patent expiration, the commercial launch of generic gemcitabine has been licensed to Teva Pharmaceuticals. According to IMS Health, the annual U.S net sales of Gemzar were nearly $785 million. Since then, net sales have dropped ~72% to 91 million due to generic competition in most major markets. Chemically, gemcitabine (2, 2 -difluorodeoxycytidine, dfdc), has a molecular weight of g/mol and is water-soluble ( g/l). Its structure, like many drugs in the anti-metabolite class, is closely related to the analogue of deoxycytidine, which fluoride replace the hydrogen atoms on the 2 carbons of the deoxycytidine. Literature reports that gemcitabine acts on the G1, M, and G2 cell cycle phase by replacing deoxycytidine during DNA replication, thus interrupting cell division, inhibiting DNA synthesis, and inducing programmed cell death [7-9]. Gemcitabine is considered a pro-drug, thus, in order for gemcitabine to exert its therapeutic action, the molecule must first traverse the cell membrane and be activated [7]. However, since gemcitabine is water-soluble and cannot pass through the cell membrane via passive diffusion, specialized transport systems, known as nucleoside transporters (i.e human equilibrative nucleoside transporter-1), are required for both 4

26 cellular entry and exit. Upon cell entry, gemcitabine is phosphorylated by deoxycytidine kinase (dck) into gemcitabine monophosphate. It is then further phosphorylated into gemcitabine diphosphate and gemcitabine triphosphate [7]. The triphosphate derivative is then intercalated into DNA by DNA-polymerase alpha to inhibit DNA synthesis and induce programmed cell death. In addition, the diphosphate derivative acts as ribonucleotide reductase inhibitor that increases gemcitabine s DNA incorporation. 1.4 Gemcitabine and its Clinical Limitations Patients with advanced pancreatic adenocarcinoma are considered incurable with a median survival of less than one year [10-11]. The gold standard treatment is a single agent, gemcitabine. Despite its favorable in vitro toxicity profiles, the therapeutic efficacy of gemcitabine as a single agent is not clinically efficacious due its rapid in vivo metabolism (i.e short half-life of minutes for short infusions in humans), preclinical studies revealing patient tolerability is highly dose dependent, and induction of tumor-drug resistance [12-13]. For example, its reported that about ninety percent of the active gemcitabine triphosphate is rapidly eliminated in vivo due to the deamination to an inactive metabolite 2`, 2`-difluorodeoxyuridine (dfdu) by cytidine deaminase [14]. By synthesizing an amino acid ester prodrug of gemcitabine, Bergman et al. demonstrated gemcitabine s in vivo sensitivity to deamination by cytidine deaminase was significantly improved. Gemcitabine is water-soluble and must be intracellularly activated, thus membrane transporters are needed to enter tumor cells as mentioned previously in this chapter. Immordino et al. synthesized a series of increasingly lipophilic gemcitabine derivatives by linking the 4-amino group with valeroyl, heptanoyl, lauroyl, and stearyol linear acyl fatty acid groups. They report that the lipophilic gemcitabine derivatives (90% recovered) degraded much slower in plasma than gemcitabine (40% recovered) after 8 5

27 hours. Furthermore, when gemcitabine or the lipophilic derivative of gemcitabine (i.e stearoyl gemcitabine) was intravenously administered to mice and their pharmacokinetic parameters were evaluated, the terminal half-life of gemcitabine was increased by 3.5 times to that of unmodified gemcitabine (0.54 h vs h, respectively) [15-16]. It may seem when the metabolic problems of gemcitabine are addressed and circumvented, the anti-tumor activity would improve as well. Surprisingly, when Zhu et al. evaluated the in vivo anti-tumor activity of a stearoyl gemcitabe solution in a B16-F10 tumor model after intravenous injection, no significant differences were observed between stearoyl gemcitabine, unmodified gemcitabine, and normal saline. 1.5 Cancer Nanotechnology During the development and growth of solid tumors, a process known as angiogenesis is induced to accommodate tumor dependency for nutrients and oxygen to grow beyond 1-2 mm in size [2]. Unlike blood vessels in normal tissue, tumor vessels become irregular in shape, dilated, and leaky with large fenestrations [17]. In addition, tumor blood vessels develop wide lumens and have impaired lymphatic drainage, which drives the extensive leakage of blood into the tumor tissue. As a result, the structural defects provide potential to propel macromolecules or nanoparticles to extravasate through gaps in the tumor vasculature and accumulate due to the slow venous return in tumor tissues and poor lymphatic drainage [17]. Exploiting this tumor biological phenomenon is known as the Enhanced Permeation and Retention (EPR) effect. An increasing emergence of nanotechnology has made a significant impact for cancer therapy by enhancing therapeutic effectiveness, reducing adverse side effects and improving drug pharmacokinetics [18]. In regards to size, the National Nanotechnology Initiative defines nano-scaled technology in the dimensions of 1 to 1000 nanometers. As 6

28 previously mentioned, nanoparticles with prolonged circulation time have the ability to accumulate at the tumor site via the EPR effect. For example, a 10- to 50-fold increase in drug accumulation was achieved using nanoparticles over nanoparticle-free drug after intravenous injection [19]. This observed phenomenon is referred to as Passive targeting, making nanoparticle-based therapeutics a promising delivery system for intravenous cancer therapy. It s important to note that the extent and rate of tumor drug accumulation using nanoparticles is largely influenced by particle size, stability, shape, surface characteristics, as well as in vivo blood circulation time. For example, nanoparticles are commonly engineered with poly-ethylene glycol (PEG) to reduce the uptake by the reticuloendothelial system and increase blood circulation time versus non- PEG counterparts [17, 20-21]. Presently, there are several FDA approved nanoparticle-based drug products, as well as several in various stages of clinical trials [22]. To name a few, doxorubicin was formulated into liposomes, commercially known as Doxil, for the treatment of patients with ovarian cancer; and paclitaxel-bound protein nanoparticles, known as Abraxane, for the treatment of metastatic breast cancer. The FDA approved Doxil and Abraxane in 1999 and 2005, respectively [22]. However, there are currently no FDA approved products that incorporate gemcitabine into nanoparticles. There are several research groups attempting to optimize the in vivo properties of gemcitabine using nanoparticle delivery systems [15-16,23-24]. For example, gemcitabine has been covalently coupled with 1,1`,2-tris-nor-squaenic acid to formulate 4-(N)-Tris-nor-squalenoyl-gemcitabine (SQdFdC NA). Following intravenous treatment in murine metastatic leukemia L1210 wt bearing mice, the SQdFdC Na caused significant increase in mouse survival time as compared to gemcitabine alone [24]. After synthesis of stearoyl gemcitabine and incorporation into liposomes, Immordino et al. observed that 7

29 the in vivo anti-tumor activity and pharmacokinetic properties were significantly enhanced versus liposome-free stearoyl gemcitabine. 1.6 Cancer Drug Targeting: The Epidermal Growth Factor Receptor Prior to FDA approval of any cancer drug, the main objective is to provide evidence of clinical effectiveness through significant survival return, beneficial effects to cancer-related symptoms and improved patient quality of life [25]. Ultimately, the beneficial effects of cancer treatment should outweigh any potential associated toxic or adverse clinical endpoints. Simply put, the performance of any drug is measured by its ability to achieve therapeutic benefit without damaging healthy, living cells. As previously mentioned in this chapter, normal and tumor cells both involve the cell cycle and undergo cell proliferation as a biological innate characteristic. It is well known that traditional chemotherapeutic drugs tend to exhibit deleterious effects on normal cells as well, particularly those with speedy turnover such as bone marrow cells, red blood cells, mucous membrane cells, hair follicle cells, and reproductive cells [2,17]. These cells are highly vulnerable, causing common clinically toxic effects like bone marrow depression, low white blood count (i.e. prone to infections), anemia, fatigue, vomiting, hair-loss, diarrhea, and fertility changes, to name a few. These foreseen and undesirable side effects are mainly due to the lack of drug specificity of the chemotherapeutics and encourage most chemotherapeutics to be used near their maximum tolerated dose. In addition, many of the chemotherapeutic drugs are low molecular weight molecules and are excreted easily by the body [26]. This remains one of the major limitations in cancer treatment. Therefore, the development of tumor-specific cytotoxic effects is highly sought after. A large body of research has accumulated demonstrating efforts to increase tumor-drug specificity can be achieved by means of active targeting [27]. 8

30 Over 100 years ago, Paul Ehrlich cleverly referenced the term, magic bullet after screening and connecting Salvarsan as the first selective and host friendly treatment option for Treponema pallidum, the causative agent for syphilis [17]. Prior to the discovery of salvarsan, targeted therapy for syphilis has not been achieved and highly toxic trivalent arsenic compounds were used. Since then, the term, active targeted therapy, has been used to describe the concept of overcoming therapeutic limitations poised by intravenously (parental) administered drugs. In cancer therapy, chemotherapeutics can be tumor targeted though a variety of means such as physical, biological, or molecular systems by taking advantage of tumor associated receptors including the epidermal growth factor receptor (EGFR) [27]. Epidermal growth factor (EGF) is a ligand to the EGFR, a 170 kd endogenous cell surface glycoprotein that, when stimulated, acts through protein-tyrosine kinase activity to stimulate several normal cellular functions including cell proliferation, survival, adhesion, migration, and differentiation. In addition to EGF, more than ten ligands have been identified to exhibit strong receptor-ligand affinity towards EGFR [27-28]. Epidermal growth factor receptor is expressed in a variety of normal cell types and there are many reports showing that EGFR is overexpressed in several tumor cells ( times greater than normal cell expression of 1 x 10 4 receptors per cell), including those cell types where gemcitabine hydrochloride is indicated for usage [28-29]. For example, it has been discovered that EGFR is over-expressed in 30-50% of pancreatic cancer cells, % of human head and neck cancer cells, and 14-91% of human breast cancer cells [29]. Multiple EGFR targeting agents have been developed for cancer therapy, and the clinical efficacy are those EGFR targeting agents have been promising. For example, EGF and antibodies against EGFR had been conjugated onto liposomes and targeted to EGFR over-expressing tumor cells [30-31]. Similarly, Arya et al. (2011) 9

31 reported that conjugation of anti-her2, an antibody against human EGFR-2, onto a gemcitabine-chitosan nanoparticle enhanced the anti-proliferative activity of the nanparticles against HER2-expressing Mia PaCa-2 cells and PANC-1 tumor cells in culture. 10

32 1.7 References 1. American Cancer Society (2012). Cancer Facts & Figures Atlanta: American Cancer Society. 2. Rubin, R. and D. Strayer (2008). Rubin's Patholoty: Clinicopathologic Foundations of Medicine. Philadelphia, Lippincott Williams & Wilkins. 3. DiPiro, J. T., R. L. Talbert, et al. (2008). Pharmacotherapy: A Pathophysiologic Approach. New York, McGraw-Hill Medical. 4. Jain, R. K. (1996). "Delivery of molecular medicine to solid tumors." Science 271(5252): Collins, K., T. Jacks, et al. (1997). "The cell cycle and cancer." Proc Natl Acad Sci U S A 94(7): Priestman, T. (2008). Cancer therapy in clinical practice. London, Springer-Verlag. 7. Abbruzzese, J. L., R. Grunewald, et al. (1991). "A phase I clinical, plasma, and cellular pharmacology study of gemcitabine." J Clin Oncol 9(3): Reid, J. M., W. Qu, et al. (2004). "Phase I trial and pharmacokinetics of gemcitabine in children with advanced solid tumors." J Clin Oncol 22(12): Huang, P., S. Chubb, et al. (1991). "Action of 2',2'-difluorodeoxycytidine on DNA synthesis." Cancer Res 51(22): Philip, P. A. (2010). "Novel targets for pancreatic cancer therapy." Surg Oncol Clin N Am 19(2): Lillemoe, K. D. (1995). "Current management of pancreatic carcinoma." Ann Surg 221(2): Castelli, F., M. G. Sarpietro, et al. (2007). "Interaction of lipophilic gemcitabine prodrugs with biomembrane models studied by Langmuir-Blodgett technique." J Colloid Interface Sci 313(1): Barton-Burke, M. (1999). "Gemcitabine: a pharmacologic and clinical overview." Cancer Nurs 22(2):

33 14. Bouffard, D. Y., J. Laliberte, et al. (1993). "Kinetic studies on 2',2'- difluorodeoxycytidine (Gemcitabine) with purified human deoxycytidine kinase and cytidine deaminase." Biochem Pharmacol 45(9): Brusa, P., M. L. Immordino, et al. (2007). "Antitumor activity and pharmacokinetics of liposomes containing lipophilic gemcitabine prodrugs." Anticancer Res 27(1A): Immordino, M. L., P. Brusa, et al. (2004). "Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing lipophilic gemcitabine prodrugs." J Control Release 100(3): Sinko, P. J. and A. N. Martin (2006). Martin's physical pharmacy and pharmaceutical sciences: physical chemical and biopharmaceutical principles in the pharmaceutical sciences.. Philadelphia, Lippincott Williams & Wilkins. 18. Zhang, L., F. X. Gu, et al. (2008). "Nanoparticles in medicine: therapeutic applications and developments." Clin Pharmacol Ther 83(5): Cuenca, A. G., H. Jiang, et al. (2006). "Emerging implications of nanotechnology on cancer diagnostics and therapeutics." Cancer 107(3): Owens, D. E., 3rd and N. A. Peppas (2006). "Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles." Int J Pharm 307(1): Allen, C., N. Dos Santos, et al. (2002). "Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol)." Biosci Rep 22(2): Wang, A. Z., R. Langer, et al. (2012). "Nanoparticle delivery of cancer drugs." Annu Rev Med 63: Stella, B., S. Arpicco, et al. (2007). "Encapsulation of gemcitabine lipophilic derivatives into polycyanoacrylate nanospheres and nanocapsules." Int J Pharm 344(1-2): Arias, J. L., L. H. Reddy, et al. (2008). "Magnetoresponsive squalenoyl gemcitabine composite nanoparticles for cancer active targeting." Langmuir 24(14): O'Shaughnessy, J. A., R. E. Wittes, et al. (1991). "Commentary concerning demonstration of safety and efficacy of investigational anticancer agents in clinical trials." J Clin Oncol 9(12):

34 26. Bharali, D. J., M. Khalil, et al. (2009). "Nanoparticles and cancer therapy: a concise review with emphasis on dendrimers." Int J Nanomedicine 4: Byrne, J. D., T. Betancourt, et al. (2008). "Active targeting schemes for nanoparticle systems in cancer therapeutics." Adv Drug Deliv Rev 60(15): LeMaistre, C. F., C. Meneghetti, et al. (1994). "Targeting the EGF receptor in breast cancer treatment." Breast Cancer Res Treat 32(1): Klijn, J. G., P. M. Berns, et al. (1992). "The clinical significance of epidermal growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients." Endocr Rev 13(1): Baselga, J. (2000). "Monoclonal antibodies directed at growth factor receptors." Ann Oncol 11 Suppl 3: Ranson, M., L. A. Hammond, et al. (2002). "ZD1839, a selective oral epidermal growth factor receptor-tyrosine kinase inhibitor, is well tolerated and active in patients with solid, malignant tumors: results of a phase I trial." J Clin Oncol 20(9):

35 Chapter 2: In Vivo and In Vitro Anti-tumor Activities of a Gemcitabine Derivative Carried by Nanoparticles Contributing authors: Brian R. Sloat, Dong Li, Woon-Gye Chung, Dharmika Lansakara-P, Philip Proteau, Kaoru Kiguchi, John J. DiGiovanni, and Zhengrong Cui. Published in and adopted from: International Journal of Pharmaceutics, 409 (2011), pp Abstract Gemcitabine (Gemzar ) is the first line treatment for pancreatic cancer and often used in combination therapy for non-small cell lung, ovarian, and metastatic breast cancers. Although extremely toxic to a variety of tumor cells in culture, the clinical outcome of gemcitabine treatment still needs improvement. In the present study, a new gemcitabine nanoparticle formulation was developed by incorporating a previously reported stearic acid amide derivative of gemcitabine into nanoparticles prepared from lecithin/glyceryl monostearate-in-water emulsions. The stearoyl gemcitabine nanoparticles were cytotoxic to tumor cells in culture, although it took a longer time for the gemcitabine in the nanoparticles to kill tumor cells than for free gemcitabine. In mice with pre-established model mouse or human tumors, the stearoyl gemcitabine nanoparticles were significantly more effective than free gemcitabine in controlling the tumor growth. PEGylation of the gemcitabine nanoparticles with polyethylene glycol (2000) prolonged the circulation of the nanoparticles in blood and increased the accumulation of the nanoparticles in tumor tissues (>6-fold), but the PEGylated and un- PEGylated gemcitabine nanoparticles showed similar anti-tumor activity in mice. Nevertheless, the nanoparticle formulation was critical for the stearoyl gemcitabine to 14

36 show a strong anti-tumor activity. It is concluded that for the gemcitabine derivatecontaining nanoparticles, cytotoxicity data in culture may not be used to predict their in vivo anti-tumor activity, and this novel gemcitabine nanoparticle formulation has the potential to improve the clinical outcome of gemcitabine treatment. 2.2 Introduction Gemcitabine (2, 2 -difluorodeoxycytidine, dfdc) is the active ingredient in Gemzar (Eli Lilly & Co., Indianapolis, IN), which is the first line treatment for pancreatic cancer [1]. The therapeutic efficacy of Gemzar as a single agent is modest, and thus, Gemzar is often used in combination therapy for non-small cell lung cancer, ovarian cancer, and metastatic breast cancer. Although extremely cytotoxic to tumor cells in culture, the clinical efficacy from gemcitabine (Gemzar ) treatment requires further improvement [2,3]. Gemcitabine is a prodrug, and its mechanism of action is based solely on intracellular phosphorylation into its active triphosphate derivative [4]. About ninety percent of gemcitabine triphosphate (dfdctp) is rapidly eliminated, mainly due to deamination to 2, 2 -difluorodeoxyuridine (dfdu), a gemcitabine derivative with minimal anti-tumor activity [5]. The rapid metabolism of gemcitabine explains its short half-life (32-84 min for short infusions in humans) [6,7,8] and is thought to be responsible for its modest clinical activity [6]. Consequently, alternative methods were explored to improve the gemcitabine formulation such as enhancing the lipophilicity of gemcitabine by conjugating long fatty acid chains onto it. It was shown that a fatty acid ester derivative of gemcitabine (CP-4126, gemcitabine-5 -elaidic acid ester) exhibited a better anti-tumor activity than its parent compound when given orally or intraperitoneally to mice [9], but an intravenous formulation of the CP-4126 was not reported. It was also 15

37 shown that incorporation of a gemcitabine fatty acid amide derivative (4-(N)-stearoylgemcitabine, GemC18) into liposomes offered advantages including hindered metabolic deactivation and improved anti-tumor activity in mouse models [10,5]. Recently, nanoparticles have gained attention as a delivery system for anticancer drugs including gemcitabine [11-15]. For example, gemcitabine had been covalently coupled with 1,1,2- tris-nor-squalenic acid to formulate 4-(N)-Tris-nor-squalenoyl-gemcitabine (SQdFdC NA) [11]. Following intravenous treatment of murine metastatic leukemia L1210 wt bearing mice, the SQdFdC NA caused a significant increase in mouse survival time compared to gemcitabine alone [11]. However, an alternative and efficacious gemcitabine formulation other than Gemzar remains unavailable on the market. Previously, our group reported the preparation of solid lipid nanoparticles of nm from lecithin/glyceryl monostearate (GMS)-in-water emulsions [16-18]. Lecithins are components of cell membranes. They are included in intramuscular and intravenous injectables [19]. GMS is used in a variety of food, pharmaceutical, and cosmetic applications and is GRAS (generally regarded as safe) listed [20]. In the present study, the feasibility of using the solid lipid nanoparticles as a delivery system for gemcitabine was evaluated. In order to incorporate the hydrophilic gemcitabine into the lipophilic matrix of the nanoparticles, the previously reported 4-(N)-stearoyl gemcitabine was adopted to increase the lipophilicity of the gemcitabine [5]. Tween 20 was one of the components of the nanoparticles [17]. Although Tween 20 has short polyethylene glycol (PEG) chains, the chains may be too short to prevent or minimize the uptake of the nanoparticles by the reticuloendothelial system (RES) after intravenous injection. Therefore, the gemcitabine nanoparticles were PEGylated using a longer PEG (molecular weight, 2000), and the anti-tumor activities of the PEGylated and un-pegylated gemcitabine nanoparticles were evaluated in vitro and in vivo. 16

38 2.3 Materials and Methods Chemicals and Cell-lines Acetone, dioxane, mannitol, ethyl acetate (EtOAc), ethylchloride, dichloromethane (CH 2 Cl 2 ), anhydrous dimethylformamide (DMF), hexane, ammonium chloride (NH 4 Cl), trifluoroacetic acid (TFA), human plasma, isopropanol, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Sepharose 4B, sodium sulfate (Na 2 SO 4 ), sodium carbonate (Na 2 CO 3 ), stearic acid, sodium chloride (NaCl), 1- hydroxy-7-aza-benzotriazole (HOAt), methanol, sodium dodecyl sulfate (SDS), and 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) were from Sigma-Aldrich (St. Louis, MO). Gemcitabine hydrochloride (gemcitabine HCl) was from U.S. Pharmacopeia (Rockville, MD). Soy lecithin was from Alfa Aesar (Ward Hill, MA). GMS was from Gattefosse Corp (Paramus, NJ). Mouse lung (TC-1, ATCC # CRL-2785) and human pancreatic (BxPC-3, ATCC # CRL-1687) cancer cell lines were from American Type Culture Collection (ATCC, Manassas, VA). TC-1 cells were grown in RPMI1640 medium (Invitrogen, Carlsbad, CA). BxPC-3 cells were grown in DMEM medium (Invitrogen). All media were supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 U/mL of penicillin (Invitrogen), and 100 μg/ml of streptomycin (Invitrogen) Synthesis of 4-(N)-Stearoyl Gemcitabine (synthesis performed by Dong Li & Dharmika S.P. Lansakara-P et al.) The 4-(N)-stearoyl-gemcitabine was prepared as previously described with slight modifications [5,20]. Briefly, 3,5 -O-bis(tert-butoxycarbonyl) gemcitabine was synthesized following a literature protocol [20]. This Boc-protected gemcitabine (179 mg, 0.39 mmol), stearic acid (121 mg, 0.42 mmol), and HOAt (57 mg, 0.42 mmol) were 17

39 dissolved in 4 ml of freshly distilled CH 2 Cl 2. After the solution was cooled in an icewater bath, EDCI (89 mg, 0.46 mmol) was added. The reaction mixture was stirred under argon for 30 h. The mixture was diluted with 10 ml of water and extracted with EtOAc/hexane mixture (2:1). The combined organic phases were washed with saturated NH 4 Cl and NaCl, dried over anhydrous Na 2 SO 4, and concentrated. The crude product was purified by silica chromatography (3:7 EtOAc/hexane). The purified Boc-protected-Nstearoyl gemcitabine (230 mg, 0.32 mmol) was dissolved in 4 ml of freshly distilled CH 2 Cl 2, and 1 ml of TFA was added. The solution was stirred at room temperature for 2 h. The solvent and excess TFA were removed in vacuo to provide the desired product, which was confirmed using 1H NMR and MS data [5] Incorporation of 4-(N)-Stearoyl Gemcitabine Into Nanoparticles Nanoparticles were prepared as previously described [17]. Briefly, 3.5 mg of soy lecithin and 0.5 mg of GMS were weighed into a 7 ml glass vial. One ml of de-ionized and filtered (0.22 μm) water was added into the lecithin/gms mixture, which was then maintained on a C hot plate while stirring until a homogenous slurry was formed. Tween 20 was added drop-wise to a final concentration of 1% (v/v). The resultant emulsions were allowed to cool to room temperature while stirring to form nanoparticles. To incorporate GemC18 into the nanoparticles to form stearoyl gemcitabine nanoparticles (GemC18-NPs), a predetermined amount of GemC18 was added into the lecithin and GMS mixture before the addition of water. The remaining steps were identical to the preparation of the gemcitabine-free nanoparticles. The size and zeta potential of the nanoparticles were measured using a Malvern Zetasizer Nano ZS (Westborough, MA). To monitor the short-term stability of the GemC18-NPs, the 18